Endothelial Dysfunction — a Major Mediator of Diabetic Vascular Disease

Biochimica et Biophysica Acta 1832 (2013) 2216–2231

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Biochimica et Biophysica Acta

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Review Endothelial dysfunction — A major mediator of diabetic vascular disease

Cristina M. Sena ⁎, Ana M. Pereira, Raquel Seiça

Institute of Physiology, Faculty of Medicine, University of Coimbra, Portugal IBILI, Faculty of Medicine, University of Coimbra, Portugal article info abstract

Article history: The vascular endothelium is a multifunctional organ and is critically involved in modulating vascular tone and Received 19 February 2013 structure. Endothelial cells produce a wide range of factors that also regulate cellular adhesion, thromboresistance, Received in revised form 31 July 2013 smooth muscle cell proliferation, and vessel wall inflammation. Thus, endothelial function is important for the Accepted 20 August 2013 homeostasis of the body and its dysfunction is associated with several pathophysiological conditions, including Available online 29 August 2013 atherosclerosis, hypertension and diabetes. Patients with diabetes invariably show an impairment of endothelium-dependent vasodilation. Therefore, understanding and treating endothelial dysfunction is a major focus in Keywords: Endothelial dysfunction the prevention of vascular complications associated with all forms of diabetes mellitus. The mechanisms of endo- Endothelium thelial dysfunction in diabetes may point to new management strategies for the prevention of cardiovascular dis- Diabetes ease in diabetes. This review will focus on the mechanisms and therapeutics that specifically target endothelial Vascular function dysfunction in the context of a diabetic setting. Mechanisms including altered glucose metabolism, impaired insu- Oxidative stress lin signaling, low-grade inflammatory state, and increased reactive oxygen species generation will be discussed. Potential therapeutic target Theimportanceofdevelopingnewpharmacologicalapproaches that upregulate endothelium-derived nitric oxide synthesis and target key vascular ROS-producing enzymes will be highlighted and new strategies that might prove clinically relevant in preventing the development and/or retarding the progression of diabetes associated vascular complications. © 2013 Elsevier B.V. All rights reserved.

1. Introduction (ET-1) [11], angiotensin II and reactive oxygen species (ROS) are among the mediators that exert vasoconstrictor effects [12,13].Endo-

1.1. Vascular function and endothelium thelial cells also produce antithrombotic (NO and PGI2 both inhibit platelet aggregation) and prothrombotic molecules [von Willebrand The endothelium is a monolayer of cells covering the vascular factor, which promotes platelet aggregation, and plasminogen activator lumen. For many years this cell layer was thought to be relatively inhibitor-1 (PAI-1), which inhibits fibrinolysis] [5]. inert, a mere physical barrier between circulating blood and the under- As a major regulator of vascular homeostasis, the endothelium main- lying tissues. It is now recognized, however, that endothelial cells are tains the balance between vasodilation and vasoconstriction, inhibition metabolically active with important paracrine, endocrine and autocrine and promotion of the migration and proliferation of smooth muscle functions, indispensable for the maintenance of vascular homeostasis cells, fibrinolysis and thrombogenesis as well as prevention and stimula- under physiological conditions [1,2]. The multiple functions of vascular tion of the adhesion and aggregation of platelets (Fig. 2) [5]. Disturbing endothelium are summarized in Fig. 1 and include regulation of vessel this tightly regulated equilibrium leads to endothelial dysfunction. integrity, vascular growth and remodeling, tissue growth and metabolism, immune responses, cell adhesion, angiogenesis, hemostasis and vascular permeability. The endothelium plays a pivotal role in the regu- 1.2. Nitric oxide lation of vascular tone, controlling tissue blood flow and inflammatory responses and maintaining blood fluidity [3–5]. NO is a crucial player in vascular homeostasis. NO is synthesized with- Endothelium-derived factors with vasodilatory and antiproliferative in endothelial cells during conversion of L-arginine to L-citrulline by endo- effects include endothelium-derived hyperpolarization factor (EDHF) [], thelial nitric oxide synthase (eNOS) [15]. It is released from endothelial nitric oxide (NO) [8,9] and prostacyclin (PGI2) [10], while endothelin-1 cells mainly in response to shear stress elicited by the circulating blood or receptor-operated substances such as acetylcholine, bradykinin, or se- rotonin [16]. NO diffuses to vascular smooth muscle cells (VSMC) and activates soluble guanylate cyclase (sGC), yielding increased levels of cyclic ⁎ Corresponding author at: Institute of Physiology, Sub-unidade 1, Pólo III, Faculty of guanosine-3,5-monophosphate (cGMP) and relaxation of VSMC [1,17]. Medicine, University of Coimbra, Azinhaga de Santa Comba, Celas, 3000-504 Coimbra, Portugal. Tel.: +351 239 480013; fax: +351 239 480034. Additionally, NO also prevents leukocyte adhesion and migration, smooth E-mail address: [email protected] (C.M. Sena). muscle cell proliferation, platelet adhesion and aggregation, and opposes

1.2.1. Decreased formation of NO White cell trafficking eNOS is a dimeric enzyme depending on multiple cofactors for its Vascular physiological activity and optimal function. eNOS resides in the caveolae Thrombosis permeability and is bound to the caveolar protein, caveolin-1 that inhibits its activity. Elevations in cytoplasmic Ca2+ promote binding of calmodulin to eNOS that subsequently displaces caveolin and activates eNOS [22,23]. eNOS utilizes L-arginine as the substrate, and molecular oxygen and Platelet Vascular tone reduced nicotinamide adenine dinucleotide phosphate (NADPH) as activation co-substrates. Flavin adenine dinucleotide, ﬂavin mononucleotide,

tetrahydrobiopterin (BH4) and calmodulin are the cofactors [4]. A re- Endothelium duced expression and/or activity of eNOS could be responsible for a decrease in NO production. Oxidative stress leads to eNOS uncoupling, a process where eNOS is converted from an NO-producing enzyme to an − Fibrinolysis Angiogenesis enzyme that generates superoxide anion (O2• ). Mechanisms implicated in eNOS uncoupling include oxidation of BH4 (a critical eNOS cofactor; [24]), depletion of the enzyme substrate L-arginine, and accumulation of endogenous methylarginines [25]. More recently, S-glutathionylation VSMC of eNOS has also been proposed as a mechanism that leads to eNOS Inflammation proliferation uncoupling and decreased NO bioavailability [26]. Additionally, increased expression of caveolin-1 in the endothelium (as described in di- Metabolism abetes and obesity [190]) leads to impaired activation of eNOS. eNOS activity within the endothelial cell is also modulated by circulating factors like insulin. Insulin is an essential hormone in Fig. 1. Multiple functions of endothelium. metabolic homeostasis with a vasodilator action exerted through the phosphatidylinositol-3 kinase (PI-3K)/AKT pathway-dependent eNOS activation [27]. Insulin can modulate eNOS activity by increas-

ing BH4 synthesis [28]. Insulin-stimulated endothelial dependent va- apoptosis and inflammation having an overall antiatherogenic effect sodilatation is impaired in insulin resistance [29,18]. Conversely, (Fig. 3) [18]. eNOS plays a major role in the regulation of insulin sensitivity due The half-life of NO is very short (less than 4 s). It is rapidly metabo- to the functions of NO in peripheral tissues [30]. Previous studies lized to nitrite and then to nitrate before being excreted in the urine [4]. have shown that mice lacking eNOS are more likely to develop insu- Alternatively, NO can also be an endocrine vasoregulator, modulating lin resistance [227]. Apparently, modulation of eNOS phosphorylation blood flow in the microcirculation [19]. Importantly, reduced eNOS ex- in mice is sufficient to affect systemic insulin sensitivity indicating pression and/or NO bioavailability is associated with endothelial dys- that eNOS phosphorylation may be a novel target for the treatment of function [20,21]. insulin resistance [228].

Fig. 2. Endothelial cells are responsible for a number of physiological functions, including: 1) regulation of vascular tone through balanced production of vasodilators and vasoconstrictors; 2) control of blood fluidity and coagulation through production of factors that regulate platelet activity, the clotting cascade, and the fibrinolytic system; and 3) regulation of inflammatory processes through expression of cytokines and adhesion molecules. ACh, acetylcholine; ATR, angiotensin-II receptor; BK, bradykinin; EDHF, endothelium-derived hyperpolarization factor; − NO, nitric oxide; PAI-1, plasminogen activator inhibitor-1; PGH2, prostaglandin H2;PGI2, prostacyclin; O2• , superoxide anion; t-PA, tissue plasminogen activator; TM, thrombomodulin;

TxA2, thromboxane A2; vWF, von Willebrand factor. 2218 C.M. Sena et al. / Biochimica et Biophysica Acta 1832 (2013) 2216–2231

Fig. 3. Atheroprotective properties of nitric oxide generated by endothelial nitric oxide synthase.

− eNOS may be inhibited by endogenous products of arginine metab- the vasculature (Fig. 4). O2• readily reacts with NO to form peroxynitrite − olism such as asymmetric dimethyl-L-arginine (ADMA) [1]. Following (ONOO ), reducing NO bioavailability and contributing to impaired vaso- oxidative stress or angiotensin II administration, the observed elevation relaxation [39]. Fig. 5 shows an increase in nitrotyrosine staining in the in ADMA levels reduces NO formation and leads to endothelial dysfunc- aorta of a type 2 diabetic animal model, indicative of peroxynitrite formation. Indeed, in several prospective studies, ADMA has been noted to be tion. Additionally, lipid peroxyl radicals react with NO at almost diffusion- an independent predictor of cardiovascular events [33–35]. limited rates and may be a source of NO inactivation [40]. Also, oxidized Another factor that regulates eNOS activity in the setting of metabol- low-density lipoprotein (LDL) cholesterol may react with endothelial ic disease is adropin, which was recently recognized to be an important NO before it reaches the vascular smooth muscle cell and therefore re- regulator of energy homeostasis and insulin sensitivity. Lovren and col- duce total NO-mediated vasodilation [41]. leagues [36] demonstrated that adropin is expressed in endothelial cells and improves angiogenesis-related responses via activation of Akt, eNOS, and extracellular signal regulated kinase 1/2. Like adiponectin 1.3. Prostacyclin and leptin, adropin may be an endocrine factor that inﬂuences both insulin resistance and endothelial functions such as vasodilation and PGI2 is the major metabolite of arachidonic acid produced by cyclo- angiogenesis [36]. oxygenase in the endothelium. PGI2 activates adenylate cyclase, leading to increased production of cyclic AMP and VSMC vasodilation. Addition-

ally, PGI2 is a potent antiproliferative agent in vascular smooth muscle 1.2.2. Accelerated breakdown of NO cells, and it reduces oxidative stress and prevents cellular adhesion to

Accelerated degradation of NO by ROS is probably the major mecha- the vascular wall [42].PG12 also inhibits platelet aggregation. Clinical nism impairing NO bioavailability in states of cardiovascular disease and experimental models of diabetes are associated with decreased se- − [37,38]. In a diabetic milieu, an increment in O2• levels is observed in cretion of PGI2 [3,27].

L L

Wistar GK

L L

Wistar GK

Fig. 4. In situ detection of superoxide in arterial vessels of normal Wistar (left panels) and diabetic Goto–Kakizaki rats (GK, right panels). Superoxide production was detected as red fluo- rescence after incubation with dihydroethidium. Representative fluorescent staining of superoxide with dihydroethidium in the abdominal aorta (upper panels) and kidney arterial ves- − sels (lower panels). O2• formation significantly increased in diabetic animals when compared to age-matched controls. C.M. Sena et al. / Biochimica et Biophysica Acta 1832 (2013) 2216–2231 2219

Wistar GK

Fig. 5. Nitrotyrosine (3-NT) immunoreactivity increases in arterial vessels from diabetic Goto–Kakizaki (GK) rats. Immunoﬂuorescence staining for 3-NT (green) in aortic (upper panels) and kidney arterial sections (lower panels) isolated from Wistar (left panels) and diabetic GK (right panels) rats. Nuclei were counter-stained with DAPI (blue). The extent and intensity of immunoﬂuorescence for nitrotyrosine was much greater in arterial rings from diabetic rat animals when compared to age-matched controls. Measuring NT levels is thought to be a reliable index to analyze peroxynitrite formation.

1.4. Endothelium derived hyperpolarizing factor Numerous risk factors directly contribute to endothelial dysfunction. Some of the more important are: elevated LDL cholesterol and oxidized There are smaller arteries in which endothelium-mediated vasodila- LDL; low high-density lipoprotein (HDL) cholesterol; elevated triglycer- tion is predominately affected by endothelium-dependent hyperpolari- ides; hypertension; elevated C-reactive protein (CRP) and circulating li- zation of vascular smooth muscle cells. The mechanism partially poprotein-associated phospholipase A2 (Lp-PLA 2 — aspecific marker of responsible for the endothelium-dependent vasodilation of these arter- vascular inflammation); hyperglycemia; elevated omega-6:omega-3 ies, which persists in the presence of inhibitors of eNOS and prostacyclin, ratio [58]; hyperinsulinemia; elevated homocysteine levels; increased was first attributed to a non-characterized endothelial factor termed fibrinogen and PAI-1; smoking; insufficient vitamin D; among others EDHF [43–45]. The relative importance of the EDHF mediated mecha- [59–61]. nisms to NO mediated mechanisms alters with vessel size [46].NOis The presence of endothelial dysfunction has been implicated in the an important endothelium-dependent mediator of vascular tone in rela- pathogenesis of atherosclerosis and thrombosis, both for the loss of its tively large arteries and larger arterioles. At the level of the aorta, reduced protective capability and for the induction of proatherothrombotic NO bioavailability is proposed to be the main marker for endothelial dys- mechanisms. The major features associated with endothelial dysfunc- function. In resistance arteries, NO, prostacyclins and EDHFs are thought tion are summarized in Table 1. to be involved in mediating endothelial function [47]. Alterations in EDHF-mediated responses have been reported in diabetes [48]. Interest- 2.1. The impact of diabetes on the vasculature ingly, EDHF synthase/cytochrome P450 epoxygenase is also a source of superoxide anion [52]. Diabetes is not only a metabolic disease but also considered as a vascular disease because of its effect on macro and microcirculation of 2. Endothelial dysfunction many vascular beds. The link between diabetes and an increased incidence of cardiovascular disease is well established [62,63]. Recent evi- In the earliest stages, the principal endothelial alteration is merely dence shows that etiopathogenesis of endothelial dysfunction differs functional. Functional impairment of the vascular endothelium is in types 1 and 2 diabetes [64]; it is present at the earliest stages of met- found in all forms of cardiovascular disease [3,12] and also in people abolic syndrome and insulin resistance, and may precede the clinical di- with insulin resistance, obesity and type 2 diabetes [18]. The hallmark agnosis of type 2 diabetes by several years [65]. of endothelial dysfunction is the impaired NO bioavailability. Addition- The metabolic milieu in diabetes (i.e. hyperglycemia, excess free ally, endothelial dysfunction is characterized by one or more of the fatty acid release and insulin resistance) induces a vicious circle of following features: reduced endothelium-mediated vasorelaxation, events in the vascular wall, involving increased endothelial dysfunction, hemodynamic deregulation, impaired fibrinolytic ability, enhanced oxidative stress, low-grade inflammation and platelet hyperactivity, in turnover, overproduction of growth factors, increased expression of ad- the early stages of diabetic disease. Thereby, activation of these systems hesion molecules and inflammatory genes, excessive generation of ROS, impairs endothelial function, augments vasoconstriction, increases in- increased oxidative stress, and enhanced permeability of the cell layer flammation, and promotes thrombosis [62,63].InFig. 6 multiple mech- [53–57]. anisms that promote atherogenesis are summarized. 2220 C.M. Sena et al. / Biochimica et Biophysica Acta 1832 (2013) 2216–2231

Table 1 2.1.1. Hyperglycemia Differences between a healthy and a dysfunctional endothelium. Besides impaired Prolonged hyperglycemia and also transient, acute hyperglycemia vasodilation (↓NO, PGI2), endothelial dysfunction is characterized by increase oxidative stress (↑ nitrotyrosine and uric acid), pro-coagulant (↑PAI-1, vWF, P-selectin), and pro- have been proven to impair endothelial function in both macro- and mi- inflammatory biomarkers (↑sICAM, sVCAM, E-selectin, CRP, TNF-alpha, IL-6, MCP-1); dec- crovascular beds in animal studies and in human subjects [67–69].Al- rement in endothelial progenitor cells and increased molecular markers of damage (circu- though the effect of intensive glycemic control on the prevention of lating endothelial cells, microparticles, MPs). macrovascular disease is less profound than on the reduction of microvascular complications [70]. Hyperglycemia causes vascular damage in different cells of the vascular wall (Table 2). The mechanisms are diverse and include: 1) increased flux of glucose and other sugars through the polyol pathway; 2) augmented intracellular formation of advanced glycation end products (AGEs); 3) increment in the expression of the receptor for AGEs (RAGE) and its activating ligands; 4) activation of protein kinase protein kinase C (PKC) isoforms; and 5) overactivation of the hexosamine pathway [74]. The common pathway is oxidative stress. ROS decreases the metabolism of glucose through glycolysis, and the flux through the alter- native polyol and hexosamine pathways is increased. Hyperglycemia induced oxidative stress [71] leads to DNA damage and activation of nuclear poly(ADP-ribose) polymerase (PARP) that in turn increases production of polymers of ADP-ribose reducing glyceraldehyde 3-phosphate dehydrogenase activity. Ultimately the levels of all upstream glycolytic CECs, circulating endothelial cells; CRP, C-reactive protein; EMPs, endothelial microparti- intermediates increase. The accumulation of glycolytic intermediates ac- cles; EPCs, endothelial progenitor cells; IL-6, interleukin-6; MPs, microparticles; NO, nitric tivates damaging mechanisms: PKC pathway, hexosamine and polyol oxide; PAI-1, plasminogen activator inhibitor 1; PGI2, prostacyclin; ROS, reactive oxygen species; sICAM, soluble intercellular adhesion molecule; sVCAM, soluble vascular cell ad- pathways and AGE formation. The overall effects of these mechanisms hesion molecule; TNF-α, tumor necrosis factor alpha; vWF, von Willebrand factor. are increased oxidative stress, apoptosis and vascular permeability [74]. Additionally, glucotoxicity induces a low-grade proinflammatory condition, due to the activation of transcription factors such as nuclear

Fig. 6. Several mechanisms that foster endothelial dysfunction and vascular damage in type 2 diabetes. Various risk factors converge on the artery (center) to promote atherogenesis under diabetic conditions. These factors include: hypertension, genetic predisposition, hyperglycemia, hyperinsulinemia, oxidative stress, advanced glycation end products (AGEs), insulin resistance and increased free fatty acids (FFAs) in circulation, lipemia, increased obesity as related to some factors which characterize life-style (sedentary, drinking, smoking and eating habits), enhanced proinflammatory and prothrombotic cytokines. Peripheral tissues are resistant to insulin action, which promotes hyperglycemia and increased levels of FFAs. In insulin resistance states, the pancreas initially tries to compensate by producing more insulin, resulting in hyperinsulinemia, itself a risk factor for angiopathy. High levels of abdominal fat present the liver with elevated levels of FFAs through the portal circulation. This excess of FFAs will lead to excess production of triglyceride (TG)-rich lipoprotein particles. Hypertriglyceridemia is accompanied by a concomitant decrease in HDL. The adipocyte can also release proinflammatory cytokines such as TNF- α, which not only have direct effects on vascular wall promoting atherogenesis, but also can elicit the production of acute phase reactants by the liver, including CRP, increased fibrinogen and PAI-1. Finally, the formation of advanced glycation end products (AGEs) from glycated macromolecules can damage vasculature through different mechanisms. VLDL, very low-density lipoprotein; TNF- α, tumor necrosis factor-α; CRP, C-reactive protein; and PAI-1, plasminogen activator inhibitor-1. C.M. Sena et al. / Biochimica et Biophysica Acta 1832 (2013) 2216–2231 2221

Table 2 Examples of mechanisms implicated in diabetic macrovascular disease.

Cellular players Mechanisms

Endothelial cells Increased reactive oxygen species Decreased NO bioavailability Increased harmful metabolites (peroxynitrite, nitrotyrosine) • Insulin resistance NF-κB activation • eNOS uncoupling Increased lipid peroxidation products • EDCF •NF-B Increased glycation (AGEs) • AGEs • Decreased NO Impaired endothelial-dependent relaxation bioavailability Monocyte-derived Increased IL1β, IL6, CD36, MCP-1 • PKC macrophages Activation of protein kinase C • Hexosamine • Stress signaling Vascular smooth muscle Increased proliferation pathway • Inflammation cells Increased migration into intima • Endothelin -1 • Sorbitol Increased matrix degradation Altered matrix components • LDL oxidation • PGIS nitration (chondroitin, dermatan sulphate proteoglycans) Increased nonenzymatic collagen glycation Increased reactive oxygen species

AGEs — advanced glycation end products; IL — interleukin; MCP-1 — monocyte chemoattractant protein-1; NF-κB — nuclear factor-kappa B; NO — nitric oxide. factor-κB(NF-κB) [74–76].NF-κB is a key mediator that regulates multiple proinflammatory and proatherosclerotic target genes in endothelial cells, VSMC, and macrophages. Activation of NF-κBleadsto Fig. 7. Endothelial dysfunction in diabetes. Prolonged exposure to hyperglycemia is the major culprit in the pathogenesis of diabetic complications, involving increased ROS and an increased production of adhesion molecules, leukocyte-attracting RNS production. Oxidative stress leads to an imbalance in the vascular homeostasis due chemokines and cytokines activating inflammatory cells in the vas- to increased vasoconstriction and impaired vasorelaxation that ultimately fosters diabetic cular wall. A prothrombotic state is generated by the increased pro- endothelial dysfunction. AGEs, advanced glycation end products; EDCF, endothelium-derived duction of lesion-based coagulants, such as tissue factor, and the contracting factors; eNOS, endothelial nitric oxide synthase; FFAs, free fatty acids; PKC, pro- κ inhibitors of fibrinolysis, such as PAI-1 (Table 1). tein kinase C; PGIS, prostacyclin synthase; NF- B, nuclear factor-kappa B; NO, nitric oxide; RNS, reactive nitrogen species; ROS, reactive oxygen species. Vascular tone and remodeling are enhanced through reduced NO [167]. and an increased activity and production of vasoconstrictors (ET-1, angiotensin II, and prostanoids [74–76]) due to postprandial increases in glucose, LDL cholesterol, and hyperinsulinemia (Fig. 7). Glucose may cells by activating NO synthase via the PI-3K pathway. In insulin resis- also activate matrix-degrading metalloproteinases, enzymes implicated tance (IR) this pathway is impaired, and the production of NO is dimin- in plaque rupture and arterial remodeling, inducing similar responses ished [89]. Instead, insulin resistance activates MAPK leading to in VSMC. Glucose may also stimulate VSMC proliferation, migration, endothelial dysfunction. Insulin stimulates production of the vasocon- and altered reactivity, for example, through renin–angiotensin activation. strictor ET-1, and increases PAI-1 and cellular adhesion molecule Elevated glucose can foster glycation of proteins, promoting forma- expression [90]. In addition to the direct effects of IR on the endotheli- tion of AGEs (Fig. 8), protein cross-linking, and ROS formation. Accumu- um, it also stimulates VSMC proliferation and migration and in adipose lation of AGEs alters the functional property of matrix components and tissue is associated with excessive release of free fatty acids (FFAs), mediates sustained cellular changes. Glycation modifies the structure of which evokes pathogenic gene expression through PKC activation and the molecules and disturbs their function and receptor recognition increased oxidative stress [93]. IR- induced excess of FFAs is essential properties. In turn, binding of AGEs to their RAGE receptor increases in- also in the development of dyslipidemia, which further promotes the tracellular enzymatic superoxide production [79,80] and promotes development of a proatherogenic lipid profile. macrophage-mediated inflammation in the vessel wall [81]. AGEs also Ultimately, insulin resistance and type 2 diabetes are associated decrease NO bioavailability and eNOS expression [82,83] and increase with low-grade inflammation being reflected in increased serum expression of ET-1 in endothelial cells [84]; therefore altering the bal- levels of tumor necrosis factor-α (TNF-α), interleukin-6, PAI-1, ET- ance between NO and ET-1 to favor vasoconstriction and endothelial 1 and high-sensitive C-reactive protein, also related to endothelial dysfunction. dysfunction [98]. Thus, accelerated formation of multiple biochemical species under hyperglycemic conditions such as nonenzymatic reactive Amadori 2.1.3. FFAs products, 3-deoxyglucosone, diacylglycerol, methylglyoxal [85],AGEs, Excessive release from adipose tissue and diminished uptake by skel- ROS, and nitrosylated species, greatly contributes to endothelial dys- etal muscles increase circulating levels of FFAs in diabetes [99,100].Acute function in diabetes. The increased oxidative stress seems to be the infusion of FFAs reduces endothelium-dependent vasodilation in animal common alteration, triggered by a type 2 diabetes milieu, in which hy- models and in humans in vivo [75,101]. perglycemia is adjoined by insulin resistance, hyperinsulinemia, and Lipotoxicity by FFAs may impair endothelial function by a number of dyslipidemia [86]. related mechanisms, including increased production of ROS, increase AGE formation and activate PKC, the hexosamine pathway, and 2.1.2. Insulin resistance proinflammatory signaling to the same extent as diabetic levels of Insulin resistance refers to a decreased ability of insulin to promote hyperglycemia. FFAs have been shown to induce ROS production in glucose uptake in skeletal muscle and adipose tissue and to suppress he- the vasculature via mitochondrial uncoupling and by increasing the patic glucose output [87]. Insulin signaling is transduced via two major expression and protein content of NADPH oxidases [74,93]. FFA- pathways: metabolic and hemodynamic effects are mediated by PI-3K induced overproduction of superoxide inactivates two important and the Ras–mitogen-activated protein kinase (MAPK) pathway is antiatherogenic enzymes: prostacyclin synthase and eNOS. ROS mainly involved in gene expression regulation, cell growth and differen- also decreases the concentration of intracellular glutathione and tiation [88]. Normally, insulin stimulates NO production in endothelial makes vasculature more prone to oxidative damage. 2222 C.M. Sena et al. / Biochimica et Biophysica Acta 1832 (2013) 2216–2231

Fig. 8. The formation of advanced glycation end products (AGEs) can involve early glucose metabolites such as glyoxal and methylglyoxal, highly reactive dicarbonyls and key precursors of AGEs.

FFA-induced ROS also activate NF-κB, which further stimulates pro- uncoupled eNOS and enzymes, such as lipoxygenase and cyclooxy- duction of other proinflammatory cytokines [103–105]. By activating genase, cytochrome P450s, peroxidases and other hemoproteins IKKα, FFA treatment impairs insulin stimulated activation of eNOS and [108] are sources of ROS. NO production in endothelial cells [106]. Activation of PKC by FFAs also results in increased serine phosphorylation of IRS-1 that leads to reduced 2.2.1.1. NADPH oxidases. NADPH oxidases are multicomponent enzymes insulin-stimulated activation of PI-3 kinase, phosphoinositide-dependent functional in membranes of various cell types including endothelial cells kinase-1, Akt, and eNOS, and culminates with impaired NO production in and smooth muscle cells. NADPH family is the predominant source of − endothelium [102,107]. Ultimately, FFAs stimulate endothelial apoptosis, O2• in the human vasculature [117,119]. Of the seven Nox isoforms augment vascular oxidative stress, reduce NO bioavailability, enhance discovered, only Nox1, Nox2, Nox4 and Nox5 are expressed in blood endothelial and monocyte activation and increase inflammation [32]. vessels, with different cell-specific expressions, mode of activations The activation of metabolite sensitive pathways of vascular damage and functions (for a review see [110,112]). by increased FFA flux from insulin resistant visceral adipocytes to arte- Activation of NADPH oxidases in the vasculature occurs in response rial endothelial cells may be the metabolic link between insulin resis- to angiotensin II, other vasoactive hormones (e.g., ET-1), growth factors tance and macrovascular disease [74,94]. Increased oxidation of fatty (e.g., transforming growth factor-β), cytokines, and mechanical stimuli acids, derived in part from insulin resistance leads to oxidative stress (shear stress and stretch), among others [121,110]. Evidences from the in diabetic macrovasculature, while in diabetic microvascular disease, literature clearly point to a role of Nox isoforms in vascular disease ROS are derived mainly from intracellular hyperglycemia [91,92].In although their relative contribution remains unclear [110].Nox1and both cases, under diabetic conditions oxidative stress seems to be the common mechanism that triggers vascular dysfunction. Table 3 Approaches to access oxidative stress in biological systems. 2.2. Oxidative stress Approach Examples

Oxidative stress describes the condition wherein an excessive pro- Markers of Increase in oxidant-generating systems increased (NADPH oxidases, xanthine oxidase, mitochondrial ROS, NOS) duction of ROS overwhelms endogenous antioxidant defense mecha- pro-oxidant Direct measurements of ROS/RNS generation nisms. The resultant elevation in ROS levels has a detrimental effect activity (reduction of NBT, oxidant-sensitive dyes, direct radicals on cellular function, a consequence of ROS-induced damage to lipid measurement by ESR) membranes, enzymes and nucleic acids [108]. Markers of Low-molecular-weight antioxidants Risk factors for cardiovascular disease (CVD), including type 2 diabe- decrease in (vitamins C and E, GSH) antioxidant Antioxidant enzymes tes, are characterized by excess vascular production of ROS [108,109]. activity (SODs, GPx, GR, catalase, thioredoxin system, paraoxonase) One of the earliest consequences of oxidative stress in human subjects Total antioxidant capacity is impaired endothelium-dependent vasodilation [108]. Thus, accessing Resistant to an external oxidant oxidative stress in the vasculature could evaluate the risk for develop- Altered cellular Overall reducing activity (cyclic voltammetry) redox state GSH/GSSG ratio ment of vascular disease (Table 3). Oxidative damage Lipid oxidation (MDA, isoprostanes, 4-HNE) parameters Protein oxidation 2.2.1. Reactive oxygen species: major sources in the vasculature (protein carbonylation, S-nitrosylation, nitrotyrosine, All layers of the vascular wall have enzymatic systems capable of glutathionylation) producing ROS. ROS include the superoxide anion, the hydroxyl radical, DNA oxidation (8-hydroxydeoxyguanosine, dihydropropidium iodide) NO, lipid radicals, hydrogen peroxide (H2O2), hypochlorous acid and peroxynitrite [108]. ROS, reactive oxygen species; RNS, reactive nitrogen species; NOS, nitric oxide synthase; NADPH oxidases, nicotinamide adenine dinucleotide phosphate oxidases; NBT, nitroblue The most important sources of ROS generation in the vasculature tetrazolium; ESR, electron spin resonance; SOD, superoxide dismutase; GR, glutathione include the mitochondrial electron transport chain [114–116],NADPH reductase; GSH/GSSG, reduced glutathione/oxidized glutathione ratio; GPx, glutathione oxidases [117–119] and xanthine oxidase [108,120]. In addition, peroxidase; 4-HNE, 4-hydroxynonenal; MDA, malondialdehyde. C.M. Sena et al. / Biochimica et Biophysica Acta 1832 (2013) 2216–2231 2223

Nox2 have distinct roles in atherogenesis promoting vascular damage However, experimental investigation has provided evidence that cata- [112]. Recent data suggest an important role for Nox1 in diabetes- lase provides only moderate protection against oxidative stress [146]. associated atherosclerosis [111]. Sukumar and co-workers showed that endothelial cell-specific insulin resistance increases Nox2 expres- 2.3.3. Glutathione peroxidases − sion and leads to O2• generation in endothelial cells sufficient to foster GPxs are a family of enzymes with an important role in antioxidant arterial dysfunction [122,225]. Contrary to Nox1 and Nox2, expression defense. Like catalase, GPxs reduce H2O2 to water and lipid hydroperox- of Nox4 was recently suggested to be vasculoprotective [112,113].Ap- ides to their corresponding alcohols. Detoxification of secondary oxida- parently, under ischemia, hypertension or inflammatory stress Nox4- tion products is vital and GPxs play an important role, reducing lipid derived H2O2 was suggested to have a protective role [112,113]. Finally, peroxides [138,139,147]. Nox5 (an isoform expressed in humans but not in rodents) is also able There are several isozymes, GPx1 is the most abundant form in to generate ROS in blood vessels and seems to have a role in endothelial mammalian tissues. Mice with a disrupted GPx1 gene exhibit increased and VSMC growth [112]. susceptibility to oxidative stress-inducing agents [148], while induction of this isozyme has been shown to provide protection against oxidative 2.2.2. Endothelial nitric oxide synthase damage in endothelial cells [149]. In apoE-deficient mice, the deficiency Nitric oxide generation is dependent on eNOS homodimerization in of GPx1 accelerates and modifies atherosclerotic lesion progression the presence of BH4. However, BH4 is highly susceptible to oxidative [31,150]. Furthermore, transgenic GPx1 expression was observed to im- degradation by ONOO− and in the absence of its cofactor, eNOS fails to pair endothelial dysfunction [151]. Similarly, deficiency of GPx3 has dimerize fully, resulting in uncoupling of the enzyme and amplification been associated with decreased NO bioavailability and increased − of oxidative stress and generation of O2• rather than NO [73]. platelet-dependent thrombosis [139]. GPx4 knockout mice are not via- Uncoupled eNOS has been shown to contribute to increased superoxide ble; they die during early embryonic development. production and endothelial dysfunction in a number of CVD, including Glutathione is the principal low molecular weight, non-protein thiol coronary artery disease [129] and type 2 diabetes [130]. in the cell [138]. Mainly found in the reduced state, glutathione has numerous functions in metabolism, signal transduction and gene expres- 2.2.3. Mitochondria sion [152]. GSH acts as an electron donor and can directly scavenge

Enzymes of the inner mitochondrial membrane transfer electrons ROS but also acts as a cofactor in the conversion of H2O2 to H2Oby along the electron transport chain which generates a proton gradient, GPxs [138,139]. enabling ATP synthase to generate ATP. Under physiological conditions, Additional selenoproteins with similar antioxidant activities to GPxs this process produces ROS as byproducts [116,131,132]. Several mito- include the thioredoxins [139], while the glutathione-S-transferases chondrial antioxidant systems are in place to protect against ROS- (GSTs) are examples of nonselenocysteine containing enzymes of induced damage to mitochondrial proteins, lipids and nucleic acids. significant importance in secondary oxidative stress defense, acting to However, under conditions of oxidative stress, these antioxidant sys- detoxify reactive electrophiles [139,147]. tems are overwhelmed, allowing ROS to exert their deleterious effects and ultimately change mitochondrial function [116,131,134]. 2.3.4. Thioredoxin Thioredoxin (Trx) seems to exert most of its ROS-scavenging 2.3. Mechanisms of defense against oxidative stress properties through Trx peroxidase (peroxiredoxin), which uses endogenous SH groups as reducing equivalents. Thioredoxin is present The vasculature is endowed with protective antioxidant defense in endothelial- and vascular smooth muscle cells. Trx scavenges ROS mechanisms, both enzymatic and nonenzymatic, to counteract the det- and ONOO− and also reduce disulfides in proteins, peptides, and ox- rimental effects of ROS. Non-enzymatic antioxidant molecules include idized glutathione (GSSG) [140,153]. ascorbic acid (vitamin C), α-tocopherol (vitamin E) and glutathione, while superoxide dismutases (SODs), catalase, glutathione peroxidases 2.3.5. Heme oxygenase (GPxs) and thioredoxins represent important antioxidant enzymes Heme oxygenase (HO) has indirect antioxidant effects through which act to directly scavenge ROS, converting them to less reactive breakdownoffreehemeandtheproductionofcarbonmonoxide,as species [138–140]. well as biliverdin and bilirubin, which themselves have antioxidant properties [154]. There are two isoforms of this enzyme, a constitutive 2.3.1. Superoxide dismutases heme oxygenase, HO2, which is ubiquitously expressed in endothelial The SODs represent the first and most important line of enzymatic cells, and HO1, which is induced in response to oxidative stress, proba- antioxidant defense against ROS. A ubiquitous family of enzymes, bly as an adaptive response. There is extensive evidence that HO1 can − SODs catalyzes the conversion of O2• to H2O2 and O2 [138,139,141]. protect against vascular damage and atherogenesis [14,124,155].The Three distinct isoforms of SOD have been identified in vascular tissue: carbon monoxide has antiproliferative and anti-inflammatory as well Cu/Zn SOD (encoded by SOD1 gene) is located in the cytoplasm, as vasodilatory properties [156]. Genetic models of HO1 deficiency or MnSOD (encoded by SOD2 gene) in the mitochondria and extracellular overexpression of HO1 suggest that the actions of HO1 are important SOD (encoded by SOD3 gene). in modulating the severity of atherosclerosis [157]. The importance of SODs as an antioxidant defense mechanism has been highlighted by gene transfer studies wherein SOD overexpression 2.3.6. Paraoxonase improved endothelial function [142,143]. Overexpression of SOD2 has The paraoxonase (PON) family of enzymes acts as vascular antioxi- also been shown to prevent hyperglycemia-associated production of dant defense and protects against vascular disease [158].ThePON1 − O2• , activation of PKC and AGE formation [144], supporting a role for and PON3 enzymes are synthesized in the liver and circulate in plasma mitochondrial ROS production in diabetic macrovascular disease. associated with HDL. The capacity of HDL in decreasing HDL and LDL lipid peroxidation largely depends on its PON1 content [158]. 2.3.2. Catalase PON1 knockout mice are more prone to atherosclerosis [159] and Catalase is a highly catalytically efficient enzyme, primarily located low PON1 activity predicts acute cardiovascular events in human in peroxisomes but also functions in the cytosol and catalyzes the con- prospective studies [160]. Deletion of PON1 gene increases oxidative − version of H2O2 to water following dismutation of O2• by SOD stress in mouse macrophages [161]. PON2 is expressed in many cell [138,139,145]. Inherited catalase deficiency has been linked to elevated types. The enzyme has been shown to reduce ROS in human endo- cardiovascular risk and increased incidence of diabetes mellitus [139]. thelial cells and vascular smooth muscle cells [162].PON2-deficient 2224 C.M. Sena et al. / Biochimica et Biophysica Acta 1832 (2013) 2216–2231 mice with an apoE−/− background developed more atherosclerotic translational mechanisms involving activation of the phosphatidylinositol lesions, whereas PON2-overexpressing mice were protected against 3-kinase/protein kinase Akt pathway [172]. those lesions [163]. In addition, cGMP levels may also be increased by inhibiting its me- Diabetes is characterized by increased oxidative stress and by de- tabolism by the phosphodiesterase-5 (PDE5) enzyme. The strategy of creased PON1 activity [164]. The ability of PON1 to protect against oxi- increasing the downstream mediator cGMP without affecting NO levels dative stress involved in major diseases such atherosclerosis and may be preferred due to the mixed outcomes in stroke reported in ani- diabetes underlines the notion that strategies aimed at increasing mal models following alterations in NO levels [173]. PON1 activity and/or expression would have several benefits. sGC stimulators and activators can treat the 2 forms of sGC insuffi- ciency (i.e., diminished NO bioavailability and reduction of the catalytic 3. Potential therapeutic targets capacity of sGC). Preliminary studies with both PDE5 inhibitors and sGC-targeted drugs have shown promising results [174–176]. In type 2 diabetes, glucotoxicity, lipotoxicity, insulin resistance and a mutual interaction between these factors occur to foster the develop- 3.1.2. Therapeutic approaches to reduce oxidative stress and/or increase ment and progression of endothelial dysfunction. Conventional thera- antioxidant defense systems pies to reduce hyperglycemia, dyslipidemia and insulin resistance Given the crucial role of ROS in endothelial function, considerable represent important clinical options to improve endothelial function efforts have been made to discover therapies to reduce ROS in the vas- and delay the progression of vascular complications [165]. These con- culature. Despite promising initial observations, clinical trials with anti- ventional therapies and their effect on vascular function have been eval- oxidant vitamins C and E failed to show an improved cardiovascular uated and reviewed elsewhere [166–169]. Noteworthy, most of these outcome. Eventually new antioxidant molecules, targeted to the precise therapies are not completely effective in slowing vascular disease and locations where ROS concentrations are elevated may, at an early stage, would benefit from adjuvant cardiovascular protective therapies. inhibit the mechanisms leading to diabetic complications [177]. The ability of PON1 to protect against oxidative stress and hydrolyze 3.1. Cardiovascular therapies targeting the endothelium homocysteine thiolactone, a metabolite of homocysteine that can impair protein function promoting endothelial dysfunction, underlines The endothelium is a highly important target for therapy in cardio- the notion that strategies aimed at increasing PON1 activity and/or vascular disease [170]. It is rapidly and preferentially exposed to system- expression can be beneficial. Certain drugs (e.g. hypolipemic and ically administered agents and establishes a link with the underlying antidiabetic compounds), dietary and life-style factors (e.g. antioxidants, tissue, providing the researcher with a useful therapeutic target. polyphenols, moderate wine consumption) appear to increase PON1 activity [180,195]. Promoting an increment in PON1 activity may prove 3.1.1. Potential therapeutic options for treating endothelial dysfunction by beneficial to prevent diabetes development [229] and slow down its car- modulating eNOS diovascular complications [180,181,230]. The vascular tone of arteries is primarily controlled by the bioavail- Substances able to inhibit NADPH oxidases and prevent superoxide ability of NO, a key factor in vascular protection by preserving vessel production may be useful for treatment of endothelial dysfunction reactivity. Thus, multiple potential therapeutic targets have been identi- [224]. Several inhibitors of the NADPH oxidase have been developed to fied along the L-arginine–NOS pathway that could increase NO bioavail- specifically target NADPH oxidases with potential benefits [182–184, ability. In Fig. 9 these sites are identified and include: at the level of the reviewed in 110 and 112]. Many cardiovascular drugs interfere with substrate, L-arginine; at the level of the NO-generating enzyme, eNOS; NADPH oxidases although most likely by indirect mechanisms. Addition- at the level of the soluble guanylyl cyclase, its main target; and at the ally, flavonoids exhibit an inhibitory effect on NADPH oxidase combined − level of the main effector of NO action, cGMP [5]. with O2• scavenging [184]. Nox-signaling pathways in the vasculature Administration of NO donors such as pentaerythritol tetranitrate are likely to offer novel therapies. Discovering gene therapy targets to- (PETN) reduces oxidative stress (probably by inducing HO1) and im- wards enzymes involved in the homeostasis of vascular redox state is es- proves endothelial dysfunction [135]. Thus, diabetic patients would sential. Recently, the design and application of nanocarriers for delivery benefit greatly from organic nitrate treatment devoid of classical ad- of antioxidants to the endothelium was performed with favorable out- verse effects, such as nitrate-induced vascular oxidative stress, nitrate comes [178]. Additionally, it has been described that delivery of genes tolerance, and endothelial dysfunction (cross-tolerance) [125]. encoding antioxidant defense enzymes (e.g. superoxide dismutase, cata- NO availability can be increased augmenting NO production by lase, glutathione peroxidase, PON1 or HO1) or eNOS suppress atherogen- eNOS. The simplest way to modulate eNOS is administration of its esis in animal models [49–51,66,97,123]. Similarly, delivery of genes substrate L-arginine [126] or its essential cofactor BH4 or BH4 analogs encoding regulators of redox sensitive transcriptional factors (e.g. NF- (Figs.9,10). Folic acid and its active form 5-methyltetrahydrofolate kappa B, AP-1, and NF-E2-related factor-2) or reactive oxygen species can modulate eNOS by improving BH4 bioavailability in the vasculature scavengers has been successfully used in experimental studies [72,179]. by preventing its oxidation [218]. Midostaurin, betulinic acid and ursolic Induction of endogenous antioxidant enzymes by activators of the acid upregulate eNOS and simultaneously decrease NADPH oxidase NF-E2-related factor-2/antioxidant response element pathway may be expression. Novel small molecules AVE9488 and AVE3085 are two an interesting approach to obtain sufficient levels of antioxidants and eNOS transcription enhancers that reverse eNOS uncoupling and pre- reduce oxidative stress [72,77]. Additionally, SIRT1-mediated inhibition serve eNOS functionality and consequently increase NO bioavailability of p66Shc (a key effector driving vascular memory in diabetes) may also [136,171]. There is evidence that a cell-permeable peptide antagonizes contribute to the prevention of oxidative stress-induced endothelial the inhibitory actions of caveolin-1 on eNOS leading to increase in NO dysfunction in vascular diseases [78]. Despite the promising results production [217]. Statins, angiotensin II type 1 receptor blockers, estro- from basic science, the clinical applicability of these strategies has prov- gens and erythropoietin enhance BH4 synthesis by stimulating GTP en to be difficult and challenging. cyclohydrolase I (GCH1) expression or activity. In vivo activation of AMP-activated protein kinase (AMPK) normalizes endothelial function 3.2. Other therapeutic approaches due to an inhibition of GCH1 degradation associated with diabetes [137]. Statins, angiotensin II receptor blockers, ACE inhibitors, the aldo- Novel therapeutic approaches designed to inhibit AGE formation sterone antagonist eplerenone and the renin inhibitor aliskiren prevent and signaling (Fig. 11) [185,186],specifically directed to reduce inflam-

BH4 oxidation by decreasing the expression and/or activity of NADPH mation [187,226] and restore the ox/redox balance in the endothelium oxidase (Figs.9,10). Statins can also directly activate eNOS via post- may represent promising strategies to ameliorate vascular function in C.M. Sena et al. / Biochimica et Biophysica Acta 1832 (2013) 2216–2231 2225

Fig. 9. Potential sites of therapeutic intervention in the L-arginine–NO-synthase–soluble guanylyl cyclase pathway. They are indicated by numbers (from 1 to 14). The schematic diagram shows an endothelial cell in yellow and a vascular smooth muscle cell in light pink. The numbers indicate: (1) L-arginine supplementation. (2) Inhibition of protein arginine N- methyltransferase type I (PRMT-I) to prevent the formation of asymmetric dimethyl-L-arginine (ADMA). (3) Increasing the expression and/or the activity of dimethylarginine dimethylaminohydrolase (DDAH) to increase ADMA degradation. (4) Inhibition of arginase-2 to preclude L-arginine metabolism. (5) Increasing the expression and/or activity of endothelial nitric oxide synthase (eNOS). (6) Stimulation of endothelium-derived nitric oxide release. (7) Enhancing the expression and/or activity of guanosine triphosphate cyclohydrolase

(GCH1), to increase tetrahydrobiopterin synthesis (BH4), or direct supplementation with BH4, or with its precursors. (8) Enhancing the expression and/or activity of dihydrofolate reductase (DHFR), to increase BH4 regeneration. (9) Scavengers of reactive oxygen species (ROS). (10) Inhibition of the activity and/or expression of enzymes that generate ROS such asNADPH oxidases (NOX), cyclooxygenases (COX), lipoxygenases (LOX) or cytochrome P450 monoxygenases (P450). (11) Enhancing the expression and/or activity of enzymes that metabolized ROS such as superoxide dismutase (SOD) or glutathione peroxidase. (12) Activators of soluble guanylyl cyclase (sGC). (13) Activators of sGC. (14) Inhibitors of phosphodiesterase-5 (PDE-

5). BH2, dihydrobiopterin; CAT-1, cationic amino acid transporters; CaV, voltage-activated calcium channel; cGMP, cyclic guanosine monophosphate; FAD, flavin adenine dinucleotide; − − FMN, flavin mononucleotide; O2• , superoxide anion; ONOO , peroxynitrite; PKG, protein kinase G. diabetic state. Potential therapies also include: AMPK activators, PKC Rho-associated coiled-coil protein kinases are potential targets for inhibitors, PARP inhibitors and rho-associated coiled-coil protein kinase treatment in vascular disease as suggested by the use of specificinhibi- (ROCK) inhibitors, among others. tors as fasudil. Treatment with fasudil was protective against vascular- AMPK is recognized as a key regulator of cellular energy status that injury-induced leukocyte recruitment in wild type but not eNOS KO has favorable effects on eNOS activity, insulin sensitivity, and mitochon- mice [222]. In diabetic animal models, studies have demonstrated a sig- drial function in a variety of cell types, including vascular cells. Therefore, nificant correlation between increased RhoA activity and impaired vas- pharmacological therapeutics that activate AMPK can be an important cular function [223]. Thus, testing of fasudil and newer more specific target in treating vascular complications in diabetes [137,219]. second generation ROCK inhibitors in a diabetic setting would be of Inhibition of protein kinase C is another therapeutic approach. great interest in an effort to limit vascular complications [95]. LY333531 (ruboxistaurin mesylate) has been shown to reduce oxidative Overall it is important to identify new targets for therapy and devel- stress and inflammation by blocking PKC-β isoform activation [188]. This op new agents for clinical use. inhibitory approach may also decrease vascular insulin resistance [133]. Furthermore, chronic treatment with the PARP inhibitors in rodent 3.3. Targeting vascular disease risk factors with nutritional therapeutics models has been demonstrated to improve endothelial dysfunction associated with aging [189,191]. In addition, pharmacological inhibition Several nutritional agents such as lipoic acid, polyphenols, resvera- of PARP with PJ-34 restored endothelium-dependent vasodilation and trol, pomegranate, omega-3 fatty acids and bioavailable SOD have reduced the levels of cytokines and inflammatory response [192,193]. been shown to effectively improve and/or protect against endothelial Additionally, PARP-1 knockout protects against dyslipidemia-induced dysfunction. Indeed, a comprehensive nutritional regimen can be autonomic and vascular dysfunction in ApoE−/− mice [194].PARPin- adjoined with pharmacological approaches in order to target all of the hibitors are potential therapies for diabetic vasculopathy. Pharmacolog- risk factors that contribute to atherosclerosis. ical catalytic decomposition of peroxynitrite with FP15 has been Lipoic acid (LA) is a naturally occurring antioxidant that serves as a demonstrated to effectively eliminate peroxynitrite and prevent PARP coenzyme in energy metabolism of fats, carbohydrates, and proteins. activation both in vitro and in vivo [220,221], thereby improving cardio- It can regenerate thioredoxin, vitamin C, and glutathione, which in vascular function in various disease models. turn can recycle vitamin E. LA reduces serum glucose levels in diabetic 2226 C.M. Sena et al. / Biochimica et Biophysica Acta 1832 (2013) 2216–2231

Fig. 10. Focus on the potential eNOS-based therapeutic approaches for endothelial dysfunction. The essential NOS cofactor tetrahydrobiopterin (BH4) is synthesized from guanosine 5′- triphosphate (GTP) via a de novo pathway by the rate-limiting enzyme GTP cyclohydrolase I (GCH1). Alternatively, the synthesis of BH4 can occur via other pathways including the salvage pathway, from dihydrobiopterin (BH2)backtoBH4.Asasubstrate,L-arginine stimulates NO release from eNOS. Folic acid may improve eNOS functionality by stabilizing BH4 and stimulating the endogenous regeneration of BH2 back to BH4. Midostaurin, betulinic acid and ursolic acid upregulate eNOS and simultaneously decrease NADPH oxidase expression. AVE9488 and AVE3085 are two eNOS transcription enhancers that reverse eNOS uncoupling and improve eNOS functionality. Statins, angiotensin II type 1 receptor blockers (ARBs), estrogens and erythropoietin (EPO) enhance BH4 synthesis by stimulating GCH1 expression or activity. Statins, ARBs, angiotensin-converting enzyme (ACE) inhibitors, the aldosterone antagonist eplerenone and the renin inhibitor aliskiren prevent BH4 oxidation by decreasing the expression and/or activity of NADPH oxidase. patients [195] and improves endothelial function in subjects with met- The benefits of resveratrol include improvements in endothelial abolic syndrome [196]. In type 2 diabetic animal models, we have previ- function [204–206]. Resveratrol seems to increase the number and ously shown a reduction of endothelial dysfunction after treatment activity of endothelial progenitor cells [205]. Resveratrol benefits the with LA [21]. circulatory system by eliciting a decrease in the oxidation of LDL; by fos- Different natural polyphenols have been shown to preserve endo- tering decreases in platelet aggregation; and by promoting relaxation of thelial function and prevent cardiovascular disease. Epidemiological ev- arterioles [207]. Thus, resveratrol improves cardiovascular system by idence suggests a negative correlation between the consumption of decreasing factors that contribute to the development of atherosclerosis polyphenol-rich foods (fruits, vegetables, and cocoa contained in choc- and atherothrombosis [96,208]. olate) or beverages (wine, especially red wine, grape juice, green tea, Previous studies indicate that pomegranate and its extracts reduce among others.) and the incidence of cardiovascular disease [197–199]. oxidation and inflammation mainly through their effect on PON-1 activ- Most polyphenols are mild antioxidants, some can reduce the ity, intervening at each step in the development of atherosclerosis activity of prooxidative NADPH oxidases, and others can stimulate [209–211]. antioxidative enzymes and eNOS [200–203].Thebeneficial effects Intake of omega-3 fatty acids might reduce Lp-PLA 2 levels and of silibinin on ADMA levels and endothelial dysfunction in db/db reduce the risk of vascular disease [212,213].Omega-3fattyacids mice were recently described. The endothelium-dependent vasodi- serve as substrates for the conversion to a novel series of lipid mediators latation to ACh was impaired in db/db mice and was restored in the designated resolvins and protectins, with potent anti-inflammatory silibinin group, accompanied with a reduction of plasma and vascu- properties [187]. Studies have found that when omega-3 fatty acids lar levels of ADMA [128]. were combined with rosuvastatin or other conventional therapies, the Several molecules with antioxidant properties (such as resveratrol, combination improved endothelial function [214,215]. piceatannol, probucol, taurine) enhance dimethyl arginine dimethyl Diminished levels of the antioxidant enzyme SOD have been linked amino hydrolase activity, increasing ADMA catabolism [127]. with cardiovascular disease. Supplementation with GliSODin, a vegetal C.M. Sena et al. / Biochimica et Biophysica Acta 1832 (2013) 2216–2231 2227

Fig. 11. Potential sites of therapeutic intervention in order to reduce hyperglycemia and its downstream effects. On the left there are the potential target sites: glycemic control; glycosyl- ation inhibition; crosslink breakers; RAGE blockers; blocking of PKC signaling pathway; and blocking of apoptosis. AGEs, advanced glycation end products; MAPK, mitogenic activated protein kinase; PKC, protein kinase C; RAGE, receptor for advanced glycation end products.

SOD associated with gliadin, was effective in controlling the thick- Acknowledgements ness of the carotid artery intima and media layers as measured by ultrasonography-B [216]. Previous studies have demonstrated the We are grateful to Fundação para a Ciência e a Tecnologia (FCT, pro- preventive efﬁcacy of GliSODin at a preclinical stage in subjects jects PTDC/SAU-MET/115635/2009; Pest-C/SAU/UI3282/2011) COM- with risk factors of cardiovascular disease. PETE, QREN, Portugal for ﬁnancial support.

4. Conclusions References

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